As disclosed in U.S. Pat. No. 5,362,634, fermentation product A83543 is a family of related compounds produced by Saccharopolyspora spinosa. The known members of this family have been referred to as factors or components, and each has been given an identifying letter designation. These compounds are hereinafter referred to as spinosyn A, B, etc. The spinosyn compounds are useful for the control of arachnids, nematodes and insects, in particular Lepidoptera and Diptera species, and they are quite environmentally friendly and have an appealing toxicological profile. Tables 1 and 2 identify the structures of a variety of known spinosyn compounds:
TABLE 1 ##STR1## Factor R.sup.1' R.sup.2' R.sup.3' R.sup.4' R.sup.5' R.sup.6' R.sup.7' spinosyn A H CH.sub.3 ##STR2## (a) C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 CH.sub.3 spinosyn B H CH.sub.3 ##STR3## (b) C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 CH.sub.3 spinosyn C H CH.sub.3 ##STR4## (c) C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 CH.sub.3 spinosyn D CH.sub.3 CH.sub.3 (a) C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 CH.sub.3 spinosyn E H CH.sub.3 (a) CH.sub.3 CH.sub.3 CH.sub.3 CH.sub.3 spinosyn F H H (a) C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 CH.sub.3 spinosyn G H CH.sub.3 ##STR5## (d) C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 CH.sub.3 spinosyn H H CH.sub.3 (a) C.sub.2 H.sub.5 H CH.sub.3 CH.sub.3 spinosyn J H CH.sub.3 (a) C.sub.2 H.sub.5 CH.sub.3 H CH.sub.3 spinosyn K H CH.sub.3 (a) C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 H spinosyn L CH.sub.3 CH.sub.3 (a) C.sub.2 H.sub.5 CH.sub.3 H CH.sub.3 spinosyn M H CH.sub.3 (b) C.sub.2 H.sub.5 CH.sub.3 H CH.sub.3 spinosyn N CH.sub.3 CH.sub.3 (b) C.sub.2 H.sub.5 CH.sub.3 H CH.sub.3 spinosyn O CH.sub.3 CH.sub.3 (a) C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 H spinosyn P H CH.sub.3 (a) C.sub.2 H.sub.5 CH.sub.3 H H spinosyn Q CH.sub.3 CH.sub.3 (a) C.sub.2 H.sub.5 H CH.sub.3 CH.sub.3 spinosyn R H CH.sub.3 (b) C.sub.2 H.sub.5 H CH.sub.3 CH.sub.3 spinosyn S H CH.sub.3 (a) CH.sub.3 H CH.sub.3 CH.sub.3 spinosyn T H CH.sub.3 (a) C.sub.2 H.sub.5 H H CH.sub.3 spinosyn U H CH.sub.3 (a) C.sub.2 H.sub.5 H CH.sub.3 H spinosyn V CH.sub.3 CH.sub.3 (a) C.sub.2 H.sub.5 H CH.sub.3 H spinosyn W CH.sub.3 CH.sub.3 (a) C.sub.2 H.sub.5 CH.sub.3 H H spinosyn Y H CH.sub.3 (a) CH.sub.3 CH.sub.3 CH.sub.3 H spinosyn A 17- H CH.sub.3 H C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 CH.sub.3 Psa spinosyn D 17- CH.sub.3 CH.sub.3 H C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 CH.sub.3 Psa spinosyn E 17- H CH.sub.3 H CH.sub.3 CH.sub.3 CH.sub.3 CH.sub.3 Psa spinosyn F 17- H H H C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 CH.sub.3 Psa spinosyn H 17- H CH.sub.3 H C.sub.2 H.sub.5 H CH.sub.3 CH.sub.3 Psa spinosyn J 17- H CH.sub.3 H C.sub.2 H.sub.5 CH.sub.3 H CH.sub.3 Psa spinosyn L 17- CH.sub.3 CH.sub.3 H C.sub.2 H.sub.5 CH.sub.3 H CH.sub.3 Psa
TABLE 2 ##STR6## Factor R.sup.1' R.sup.2' R.sup.3' R.sup.4' R.sup.5' spinosyn A 9-Psa H CH.sub.3 ##STR7## (a) C.sub.2 H.sub.5 H spinosyn D CH.sub.3 CH.sub.3 (a) C.sub.2 H.sub.5 H 9-Psa spinosyn A H CH.sub.3 H C.sub.2 H.sub.5 H Aglycone spinosyn D CH.sub.3 CH.sub.3 H C.sub.2 H.sub.5 H Aglycone
The naturally produced spinosyn compounds consist of a 5,6,5-tricylic ring system, fused to a 12-membered macrocyclic lactone, a neutral sugar (rhamnose) and an amino sugar (forosamine) (see Kirst et al. (1991). If the amino sugar is not present the compounds have been referred to as the pseudoaglycone of A, D, etc., and if the neutral sugar is not present then the compounds have been referred to as the reverse pseudoaglycone of A, D, etc. A more preferred nomenclature is to refer to the pseudoaglycones as spinosyn A 17-Psa, spinosyn D 17-Psa, etc., and to the reverse pseudoaglycones as spinosyn A 9-Psa, spinosyn D 9-Psa, etc.
The naturally produced spinosyn compounds may be produced via fermentation from cultures NRRL 18395, 18537, 18538, 18539, 18719, 18720, 18743 and 18823. These cultures have been deposited and made part of the stock culture collection of the Midwest Area Northern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 1815 North University Street, Peoria, Ill. 61604.
U. S. Pat. No. 5,362,634 and corresponding European Patent Application No. 375316 A1 disclose spinosyns A, B, C, D, E, A, G, H, and J. These compounds are disclosed as being produced by culturing a strain of the novel microorganism Saccharopolyspora spinosa selected from NRRL 18395, NRL 18537, NRRL 18538, and NRRL 18539.
WO 93/09126 disclosed spinosyns L, M, N, Q, R, S, and T. Also disclosed therein are two spinosyn J producing strains: NRRL 18719 and NRRL 18720, and a strain that produces spinosyns Q, R, S, and T; NRRL 18823.
WO 94/20518 and U.S. Pat. No. 5,6704,486 disclose spinosyns K, O, P, U, V, W, and Y, and derivatives thereof Also disclosed is spinosyn K-producing strain NRRL 18743.
A challenge in producing spinosyn compounds arises from the fact that a very large fermentation volume is required to produce a very small quantity of spinosyns. It is highly desired to increase spinosyn production efficiency and thereby increase availability of the spinosyns while reducing their cost. A cloned fragment of DNA containing genes for spinosyn biosynthetic enzymes would enable duplication of genes coding for rate limiting enzymes in the production of spinosyns. This could be used to increase yield in any circumstance when one of the encoded activities limited synthesis of the desired spinosyn. A yield increase of this type was achieved in fermentations of Streptomyces fradiae by duplicating the gene encoding a rate-limiting methyltransferase that converts macrocin to tylosin (Baltz et al., 1997).
Cloned biosynthetic genes would also provide a method for producing new derivatives of the spinosyns which may have a different spectrum of insecticidal activity. New derivatives are desirable because, although known spinosyns inhibit a broad spectrum of insects, they do not control all pests. Different patterns of control may be provided by biosynthetic intermediates of the spinosyns, or by their derivatives produced in vivo, or by derivatives resulting from their chemical modification in vitro. Specific intermediates (or their natural derivatives) could be synthesized by mutant strains of S. spinosa in which certain genes encoding enzymes for spinosyn biosynthesis have been disrupted. Such strains can be generated by integrating, via homologous recombination, a mutagenic plasmid containing an internal fragment of the target gene. Upon plasmid integration, two incomplete copies of the biosynthetic gene are formed, thereby eliminating the enzymatic function it encoded. The substrate for this enzyme, or some natural derivative thereof, should accumulate upon fermentation of the mutant strain. Such a strategy was used effectively to generate a strain of Saccharopolyspora erythraea producing novel 6-deoxyerythromycin derivatives (Weber & McAlpine, 1992).
Novel intermediates could also be synthesized by mutant strains of S. spinosa in which parts of certain genes encoding enzmines for spinosyn biosynthesis have been replaced with parts of the same gene which have been specifically mutated in vitro, or with corresponding parts of genes from other organisms. Such strains could be generated by swapping the target region, via double homologous recombination, with a mutagenic plasmid containing the new fragment between non-mutated sequences which flank the target region. The hybrid gene would produce protein with altered functions, either lacking an activity or performing a novel enzymatic transformation. A new derivative would accumulate upon fermentation of the mutant strain. Such a strategy was used to generate a strain of Saccharopolyspora erythraea producing a novel anhydroeriythromycin derivative (Donadio et al., 1993).
Biosynthesis of spinosyns proceeds via stepwise condensation and modification of is 2- and 3-carbon carboxylic acid precursors, generating a linear polyketide that is cyclized and bridged to produce the tetracyclic aglycone. Pseudoaglycone (containing tri-O-methylated rhamnose) is formed next, then di-N-methylated forosamine is added to complete the biosynthesis (Broughton et al., 1991). Other macrolides, such as the antibiotic erythromycin, the antiparasitic avermectin and the immunosuppressant rapamycin, are synthesized in a similar fashion. In the bacteria producing these compounds, most of the macrolide biosynthetic genes are clustered together in a 70-80 kb region of the genome (Donadio et al., 1991; MacNeil et al., 1992; Schwecke et al., 1995). At the centers of these clusters are 3-5 highly conserved genes coding for the very large, multifunctional proteins of a Type I polyketide synthase (PKS). Together the polypeptides form a complex consisting of an initiator module and several extender modules, each of which adds a specific acyl-CoA precursor to a growing polyketide chain, and modifies the .beta.-keto group in a specific manner. The structure of a polyketide is therefore determined by the composition and order of the modules in the PKS. A module comprises several domains, each of which performs a specific function. The initiator module consists of an acyl transferase (AT) domain for addition of the acyl group from the precursor to an acyl carrier protein (ACP) domain. The extender modules contain these domains, along with a .beta.-ketosynthase (KS) domain that adds the pre-existing polyketide chain to the new acyl-ACP by decarboxylative condensation. Additional domains may also be present in the extender modules to carry out specific .beta.keto modifications: a .beta.-ketoreductase (KR) domain to reduce the .beta.-keto group to a hydroxyl group, a dehydratase (DH) domain to remove the hydroxyl group and leave a double bond, and an enoyl reductase (ER) domain to reduce the double bond and leave a saturated carbon. The last extender module terminates with a thioesterase (TE) domain that liberates the polyketide from the PKS enzyme in the form of a macrocyclic lactone.
Macrolides are derived from macrocyclic lactones by additional modifications, such as methylation and changes in reductive state, and the addition of unusual sugars. Most of the genes required for these modifications, and for the synthesis and attachment of the sugars, are clustered around the PKS genes. The genes encoding deoxysugar biosynthetic enzymes are similar in producers of macrolide antibiotics, such as erythromycin and tylosin (Donadio et al., 1993; Merson-Davies & Cundliffe, 1994), and producers of extracellular polysaccharides, such as the O-antigens of Salmonella and Yersinia (Jiang et al., 1991; Kessler et al., 1993). All these syntheses involve activation of glucose by the addition of a nucleotide diphosphate, followed by dehydration, reduction and/or epimerization. The resultant sugar could undergo one or more modifications such as deoxygenation, transamination and methylation, depending upon the type of sugar moiety present in the macrolide. The sugars are incorporated into macrolides by the action of specific glycosyltransferases. Genes involved in the synthesis and attachment of a sugar may be tightly clustered--even transcribed as a single operon-or they may be dispersed (Decker & Hutchinson, 1993; Jarvis & Hutchinson, 1994). Spinosyn synthesis also involves bridging of the lactone nucleus, an activity that is rare in macrolide producers. Therefore, the spinosyn biosynthetic cluster may uniquely contain additional genes encoding enzymes for this function.
The following terms are used herein as defined below:
AmR--the apramycin resistance-conferring gene.
ApR--the ampicillin resistance-conferring gene.
ACP--acyl carrier protein.
AT--acyltransferase.
bp--base pairs.
Cloning--the process of incorporating a segment of DNA into a recombinant DNA cloning vector and transforming a host cell with the recombinant DNA.
CmR--the chloramphenicol resistance-conferring gene.
Codon bias--the propensity to use a particular codon to specify a specific amino acid. In the case of S. spinosa, the propensity is to use a codon having cytosine or guanine as the third base.
Complementation--the restoration of a mutant strain to its normal phenotype by a cloned gene.
Conjugation--a process in which genetic material is transferred from one bacterial cell to another.
cos--the lambda cohesive end sequence.
Cosmid--a recombinant DNA cloning vector which is a plasmid that not only can replicate in a host cell in the same manner as a plasmid but also can be packaged into phage heads.
DH--dehydratase.
ER--enoyl reductase.
Exconjugant--recombinant strain derived from a conjugal mating.
Gene--a DNA sequence that encodes a polypeptide.
Genomic Library--a set of recombinant DNA cloning vectors into which segments of DNA, representing substantially all DNA sequences in a particular organism have been cloned.
Homology--degree of similarity between sequences
Hybridization--the process of annealing two single stranded DNA molecules to form a double stranded DNA molecule, which may or may not be completely base paired.
In vitro packaging--the in vitro encapsulation of DNA in coat protein to produce a virus-like particle that can introduce DNA into a host cell by infection
kb--kilo base pairs.
KR--.beta.-keto reductase.
KS--ketosynthase.
Mutagenesis--creation of changes in DNA sequence. They can be random or targeted, generated in vivo or in vitro. Mutations can be silent, or can result in changes in the amino acid sequence of the translation product which alter the properties of the protein and produce a mutant phenotype.
NmR--the neomycin resistance-conferring gene.
ORF--open reading frame.
ori--a plasmid origin of replication (oriR)or transfer (oriT).
PKS--polyketide synthase.
Promoter--a DNA sequence that directs the initiation of transcription.
Recombinant DNA cloning vector--any autonomously replicating or integrating agent, including, but not limited to, plasmids, comprising a DNA molecule to which one or more additional DNA molecules can be or have been added.
Recombinant DNA methodology--technologies used for the creation, characterization, and modification of DNA segments cloned in recombinant DNA vectors.
Restriction fragment--any linear DNA molecule generated by the action of one or more restriction enzymnes.
Spinosyn--a fermentation product typically characterized by a 5,6,5-tricylic ring system, fused to a 12-membered macrocyclic lactone, a neutral sugar (rhamnose) and an amino sugar (forosamine), or a similar macrocyclic lactone fermentation product produced by a microorganism utilizing all or most of the spinosyn genes.
Spinosyn genes--the DNA sequences that encode the products required for spinosyn biosynthesis, more specifically the genes spnA, spnB, spnC, spnD, spnE, spnF, spnG, spnH, spnI, spnJ, spnK, spnL, spnM, spnN, spnO, spnP, spnQ, spnR, spnS, S. spinosa gtt, S. spinosa gdh, S. spinosa epi, and S. spinosa kre, as described hereinafter, or functional equivalents thereof
Subclone--a cloning vector with an insert DNA derived from another DNA of equal size or larger.
TE--thioesterase.
Transformation--the introduction of DNA (heterologous or homologous) into a recipient host cell that changes the genotype and results in a change in the recipient cell.